Methodology On Biomarkers Detection Of Exposure To Lewisite

Lewisite is one kind of highly toxic Chemical Warfare Agents (CWAs) with vesicant properties, which was first synthesized by Captain Lewis during World War I (WWI). Industrial product of Lewisite is a mixture of L1 (dichloro (2-chlorovinyl) arsine, above 90 %), L2 (bis-(2-chlorovinyl)chloroarsine, about 10 %) and L3 (tris-(2-chlorovinyl)arsine, less than 1 %). Without any statement, Lewsite was represented by L1.Either direct skin contacting or breathing can lead to the exposure of Lewsite. After being absorbed, most of the Lewsite molecules bind to mercapto-beared amino acids or protein residues immediately and therefore inhibited or eliminated the bioactivities of those biomolecules by forming the"Lewsite-biomacromolecule"bioconjugates. A less percentage of Lewsite is rapidly hydrolyzed to 2-chlorovinylarsonous acid (CVAA), which also be harmful to biofunctions as a blistering agent. Both above approaches constitute the toxicological mechanism of Lewsite. Since the"Lewsite-biomacromolecule"conjugates were formed as the special bioadducts from the reactions between Lewiste and bioorganism, so they were also called as biomarkers for Lewsite. Compared with hydrolysis products, the biomarkers have the advantages of relative longer lifetime, so the detection of biomarkers can provide a direct retrospective evidence of an occupational and/or accidental exposure to L1.Based on above scientific background, a series of analytical methods were established in this thesis to verify biomarkers of L1. After the synthesis of internal standard and selection of derivatizing agents, the derivatization conditions and the recovery approaches were thoroughly investigated for urine and whole blood exposures, respectively, thus the high-sensitive analytical methods for biomedical samples were finally developed. The aim of this work is to provide a reliable support for proper medical treatments of L1 exposure. This thesis includes 5 chapters.Chapter 1 overviewed the physical, chemical and toxicological properties of L1, and more than 30 papers were cited. Accompanied with the toxicological mechanism of L1, its antidotes working pattern were also introduced. The theoretical basis was then presented for our establishing methods.In Chapter 2, an internal standard (IS) of phenylarsine oxide-dimercaprol (PAB) isomers were synthesized and purified. Several analytical tools were systematically employed to qualify and quantify this product. The purity of PAB IS is over 98 %, which meets the requirement of our analytical methods.In Chapter 3, the derivatization conditions of several mercaptan agents for L1 were optimized. The derivatizing efficiencies were compared on the best conditions of individual mercaptan agents. The aim of this research is to select suitable agents, serving for different detection methods of clinical samples or biomedical exposures. The experimental in Chapter 4 were developed for analysis of clinical exposures to L1. Firstly, the spiked water was used as a model matrix, the parameters of liquid-liquid extraction (LLE) were optimized as well as the recovery and cost between LLE and solid-phase extraction (SPE) as pretreatment techniques were compared. The organic solvents of acetic ester and dichlormethane-acetic ester (1:1, v/v) were thus selected as the most suitable solvents for extracting L1-BAL (L1-2,3-dimercapto-1-propanol) from spiked human urine and human whole blood, respectively. L1-BAL was then transferred to gas chromatography-atomic emission spectrometric detector (GC-AED). With the purpose to improve shape of chromatographic peak of analyte and enhance the sensitivity of this method, L1-BAL was further derivatized by heptafluoro-butyryl imidazole (HFBI) and consequently measured by gas chromatography-mass spectrometry (GC-MS).In the last Chapter, a high-sensitive method was established for biomedical samples, such as human urine and whole blood exposed to L1, by using toluol-3,4-dithiol (TDT) as an effective derivatization agent. Based on the optimum derivatization condition and LLE conditions, the analyte of L1-TDT (L1-toluol-3,4-dithiol) was extracted from spiked biomedical samples and determinated by GC-MS. In addition, the stability of L1-TDT in different matrixes and the transformation condition of L1-BAL in biomedical samples by TDT were investigated.The established methods for L1-BAL in this thesis can meet the need to verify clinical exposure samples. It was confirmed that BAL can be used as the antidote and derivatization agent simultaneously. In the other hand, the verification of L1-TDT in spiked samples with high-sensitive and high-retrospective can be employed for further toxicological mechanism and pratical applications. Moreover, the proposed transformation condition for L1-BAL and L1-TDT can efficiently extend the applicable ranges of such analytical methods.